The goal of this proposal is to develop a tractable model for genetic analyses of the role of host factors in Alphavirus infection. Members of the Alphavirus genus in the Togaviridae family cause significant human and animal disease, including encephalitis and death. The genus includes NIAID Category B priority pathogens of concern for possible bioterrorism use. Currently there is no specific therapy available. While host factors undoubtedly play important roles in the Alphavirus replication cycle, their identities and functions remain largely unknown. Understanding which host proteins are required for virus replication and how the virus interacts with these host proteins could open up potential targets for the development of antiviral therapies. This project will utilize the well-studied nematode Caenorhabditis elegans as a model organism for studying the role of host factors in Alphavirus replication. Techniques for mutagenesis and genetic analysis of C. elegans are well worked out. The type Alphavirus, Sindbis virus (SINV), was chosen as a test Alphavirus for model development. The first aim of the study is to generate transgenic animals expressing a GFP- expressing SINV replicon under the control of a well-characterized C. elegans promoter. SINV RNA expression will be monitored by GFP expression. In the second aim, the transgenic animals harboring the SINV replicon-encoding DNA will be subjected to chemical mutagenesis, SINV expression will be monitored, and mutants with decreased (or enhanced) SINV replication will be identified by assessment of GFP expression. Standard genetic approaches will be employed to identify the genes harboring mutations that decrease or increase SINV replication. In the third aim, the model will test for the significance in viral replication of host proteins previously identified as associating with viral replication proteins. Candidate host proteins will be compared to the genes present in C. elegans, and if available, existing mutants will be engineered (by crossing) into the SINV model strain for testing. Alternatively, the SINV model strain will be fed bacteria expressing gene-specific double stranded RNA (available for virtually all the nematode genes) to silence expression. The effect of reduced levels of the host protein on SINV replication will be assessed by GFP visualization. These studies have the potential to identify host factors necessary for Alphavirus RNA replication, which may open up new targets for possible antiviral treatment. The model has the potential to be expanded to allow examination of the entire Alphavirus life cycle through use of GFP expressing virus, rather than a replicon. In addition, the model is amenable to the study of other Alphaviruses, including NIAID Category B agents, as well as other medically important positive strand RNA viruses. PUBLIC HEALTH RELEVANCE: Viruses in the Alphavirus genus cause diseases ranging from fever, rash and arthritis, to encephalitis and death, yet to date no specific therapies exist. This study will develop a small animal (nematode) model of Alphavirus infection, which will be used to take a genetic approach to identify host factors that are required for viral replication. Understanding the virus-host interaction in greater detail may lead to the development of specific therapies.